Wild mushrooms as potential reservoirs of plant pathogenic bacteria: a case study on Burkholderia gladioli.

Fruit bodies (sporocarps) of wild mushrooms growing in natural environments play a substantial role in the preservation of microbial communities, for example, clinical and food-poisoning bacteria. However, the role of wild mushrooms as natural reservoirs of plant pathogenic bacteria remains almost entirely unknown. Furthermore, bacterial transmission from a mushroom species to agricultural plants has rarely been recorded in the literature. In September 2021, a creamy-white Gram-negative bacterial strain was isolated from the sporocarp of Suillus luteus (slippery jack) growing in Bermuda grass (Cynodon dactylon) lawn in Southern Iran. A similar strain was isolated from the same fungus in the same area in September 2022. Both strains were identified as Burkholderia gladioli based on phenotypic features as well as phylogeny of 16S rRNA and three housekeeping genes. The strains were not only pathogenic on white button mushrooms (Agaricus bisporus) but also induced hypersensitive reaction (HR) on tobacco and common bean leaves and caused soft rot on a set of diverse plant species, that is, chili pepper, common bean pod, cucumber, eggplant, garlic, gladiolus, narcissus, onion, potato, spring onion, okra, kohlrabi, mango, and watermelon. Isolation of plant pathogenic B. gladioli strains from sporocarp of S. luteus in two consecutive years in the same area could be indicative of the role of this fungus in the preservation of the bacterium in the natural environment. B. gladioli associated with naturally growing S. luteus could potentially invade neighboring agricultural crops, for example, vegetables and ornamentals. The potential role of wild mushrooms as natural reservoirs of phytopathogenic bacteria is further discussed.IMPORTANCEThe bacterial genus Burkholderia contains biologically heterogeneous strains that can be isolated from diverse habitats, that is, soil, water, diseased plant material, and clinical specimens. In this study, two Gram-negative pectinolytic bacterial strains were isolated from the sporocarps of Suillus luteus in September 2021 and 2022. Molecular phylogenetic analyses revealed that both strains belonged to the complex species Burkholderia gladioli, while the pathovar status of the strains remained undetermined. Biological investigations accomplished with pathogenicity and host range assays showed that B. gladioli strains isolated from S. luteus in two consecutive years were pathogenic on a set of diverse plant species ranging from ornamentals to both monocotyledonous and dicotyledonous vegetables. Thus, B. gladioli could be considered an infectious pathogen capable of being transmitted from wild mushrooms to annual crops. Our results raise a hypothesis that wild mushrooms could be considered as potential reservoirs for phytopathogenic B. gladioli.

Methyl jasmonate differentially and tissue-specifically regulated the expression of arginine catabolism-related genes and proteins in Agaricus bisporus mushrooms during storage.

Methyl jasmonate (MeJA)-regulated postharvest quality retention of Agaricus bisporus fruiting bodies is associated with arginine catabolism. However, the mechanism of MeJA-regulated arginine catabolism in edible mushrooms is still unclear. This study aimed to investigate the regulatory modes of MeJA on the expression of arginine catabolism-related genes and proteins in intact and different tissues of A. bisporus mushrooms during storage. Results showed that exogenous MeJA treatment activated endogenous JA biosynthesis in A. bisporus mushrooms, and differentially and tissue-specifically regulated the expression of arginine catabolism-related genes (AbARG, AbODC, AbSPE-SDH, AbSPDS, AbSAMDC, and AbASL) and proteins (AbARG, AbSPE-SDH, AbASL, and AbASS). MeJA caused no significant change in AbASS expression but resulted in a dramatic increase in AbASS protein level. Neither the expression of the AbSAMS gene nor the AbSAMS protein was conspicuously altered upon MeJA treatment. Additionally, MeJA reduced the contents of arginine and ornithine and induced the accumulation of free putrescine and spermidine, which was closely correlated with MeJA-regulated arginine catabolism-related genes and proteins. Hence, the results suggested that the differential and tissue-specific regulation of arginine catabolism-related genes and proteins by MeJA contributed to their selective involvement in the postharvest continuing development and quality retention of button mushrooms.

Classification and selection of the main features for the identification of toxicity in Agaricus and Lepiota with machine learning algorithms.

The occurrence of fungi is cosmopolitan, and while some mushroom species are beneficial to human health, others can be toxic and cause illness problems. This study aimed to analyze the organoleptic, ecological, and morphological characteristics of a group of fungal specimens and identify the most significant features to develop models for fungal toxicity classification using genetic algorithms and LASSO regression. The results of the study indicated that odor, spore print color, and habitat were the most significant characteristics identified by the genetic algorithm GALGO. Meanwhile, odor, gill size, stalk shape, and twelve other features were the relevant characteristics identified by LASSO regression. The importance score of the odor variable was 99.99%, gill size obtained 73.7%, stalk shape scored 39.9%, and the remaining variables did not score higher than 18%. Logistic regression, k-nearest neighbor (KNN), and XG-Boost classification algorithms were used to develop models using the features selected by both GALGO and LASSO. The models were evaluated using sensitivity, specificity, and accuracy metrics. The models with the highest AUC values were XGBoost, with a maximum value of 0.99 using the features selected by LASSO, followed by KNN with a maximum value of 0.99. The GALGO selection resulted in a maximum AUC of 0.98 in KNN and XGBoost. The models developed in this study have the potential to aid in the accurate identification of toxic fungi, which can prevent health problems caused by their consumption.

Unravelling the Transcriptional Response of Agaricus bisporus under Lecanicillium fungicola Infection.

Mushrooms are a nutritionally rich and sustainably-produced food with a growing global market. Agaricus bisporus accounts for 11% of the total world mushroom production and it is the dominant species cultivated in Europe. It faces threats from pathogens that cause important production losses, including the mycoparasite Lecanicillium fungicola, the causative agent of dry bubble disease. Through quantitative real-time polymerase chain reaction (qRT-PCR), we determine the impact of L. fungicola infection on the transcription patterns of A. bisporus genes involved in key cellular processes. Notably, genes related to cell division, fruiting body development, and apoptosis exhibit dynamic transcriptional changes in response to infection. Furthermore, A. bisporus infected with L. fungicola were found to accumulate increased levels of reactive oxygen species (ROS). Interestingly, the transcription levels of genes involved in the production and scavenging mechanisms of ROS were also increased, suggesting the involvement of changes to ROS homeostasis in response to L. fungicola infection. These findings identify potential links between enhanced cell proliferation, impaired fruiting body development, and ROS-mediated defence strategies during the A. bisporus (host)-L. fungicola (pathogen) interaction, and offer avenues for innovative disease control strategies and improved understanding of fungal pathogenesis.

Transcriptome and Hormonal Analysis of Agaricus bisporus Basidiome Response to Hypomyces perniciosus Infection.

Agaricus bisporus (Lange) Imbach is the most widely cultivated mushroom in the world. A. bisporus wet bubble disease is one of the most severe diseases of white button mushrooms and is caused by the fungal pathogen Hypomyces perniciosus. The pathogen causes a drastic reduction in mushroom yield because of malformation and deterioration of the basidiomes. However, the mechanism of the button mushroom's malformation development after infection with H. perniciosus remains obscure. Therefore, to reveal the mechanism of A. bisporus malformation caused by H. perniciosus, the interaction between the pathogen and host was investigated in this study using histopathological, physiological, and transcriptomic analyses. Results showed that irrespective of the growth stages of A. bisporus basidiomes infected with H. perniciosus, the host's malformed basidiomes and enlarged mycelia and basidia indicated that the earlier the infection with H. perniciosus, the more the malformation of the basidiomes. Analyzing physiological and transcriptomic results in tandem, we concluded that H. perniciosus causes malformation development of A. bisporus mainly by affecting the metabolism level of phytohormones (N6-isopentenyladenosine, cis-zeatin, and N6-[delta 2-isopentenyl]-adenine) of the host's fruiting bodies rather than using toxins. Our findings revealed the mechanism of the button mushroom's malformation development after infection with H. perniciosus, providing a reference for developing realistic approaches to control mushroom diseases. Our results further clarified the interaction between A. bisporus and H. perniciosus and identified the candidate genes for A. bisporus wet bubble disease resistance breeding. Additionally, our work provides a valuable theoretical basis and technical support for studying the interaction between other pathogenic fungi and their fungal hosts.

Decoding the chromosome-scale genome of the nutrient-rich Agaricus subrufescens: a resource for fungal biology and biotechnology.

Agaricus subrufescens, also known as the "sun mushroom," has significant nutritional and medicinal value. However, its short shelf life due to the browning process results in post-harvest losses unless it's quickly dehydrated. This restricts its availability to consumers in the form of capsules. A genome sequence of A. subrufescens may lead to new cultivation alternatives or the application of gene editing strategies to delay the browning process. We assembled a chromosome-scale genome using a hybrid approach combining Illumina and Nanopore sequencing. The genome was assembled into 13 chromosomes and 31 unplaced scaffolds, totaling 44.5 Mb with 96.5% completeness and 47.24% GC content. 14,332 protein-coding genes were identified, with 64.6% of the genome covered by genes and 23.41% transposable elements. The mitogenome was circularized and encoded fourteen typical mitochondrial genes. Four polyphenol oxidase (PPO) genes and the Mating-type locus were identified. Phylogenomic analysis supports the placement of A. subrufescens in the Agaricomycetes clade. This is the first available genome sequence of a strain of the "sun mushroom." Results are available through a Genome Browser (https://plantgenomics.ncc.unesp.br/gen.php?id=Asub) and can support further fungal biological and genomic studies.

Bacterial secretion systems contribute to rapid tissue decay in button mushroom soft rot disease.

The soft rot pathogen Janthinobacterium agaricidamnosum causes devastating damage to button mushrooms (Agaricus bisporus), one of the most cultivated and commercially relevant mushrooms. We previously discovered that this pathogen releases the membrane-disrupting lipopeptide jagaricin. This bacterial toxin, however, could not solely explain the rapid decay of mushroom fruiting bodies, indicating that J. agaricidamnosum implements a more sophisticated infection strategy. In this study, we show that secretion systems play a crucial role in soft rot disease. By mining the genome of J. agaricidamnosum, we identified gene clusters encoding a type I (T1SS), a type II (T2SS), a type III (T3SS), and two type VI secretion systems (T6SSs). We targeted the T2SS and T3SS for gene inactivation studies, and subsequent bioassays implicated both in soft rot disease. Furthermore, through a combination of comparative secretome analysis and activity-guided fractionation, we identified a number of secreted lytic enzymes responsible for mushroom damage. Our findings regarding the contribution of secretion systems to the disease process expand the current knowledge of bacterial soft rot pathogens and represent a significant stride toward identifying targets for their disarmament with secretion system inhibitors. IMPORTANCE The button mushroom (Agaricus bisporus) is the most popular edible mushroom in the Western world. However, mushroom crops can fall victim to serious bacterial diseases that are a major threat to the mushroom industry, among them being soft rot disease caused by Janthinobacterium agaricidamnosum. Here, we show that the rapid dissolution of mushroom fruiting bodies after bacterial invasion is due to degradative enzymes and putative effector proteins secreted via the type II secretion system (T2SS) and the type III secretion system (T3SS), respectively. The ability to degrade mushroom tissue is significantly attenuated in secretion-deficient mutants, which establishes that secretion systems are key factors in mushroom soft rot disease. This insight is of both ecological and agricultural relevance by shedding light on the disease processes behind a pathogenic bacterial-fungal interaction which, in turn, serves as a starting point for the development of secretion system inhibitors to control disease progression.

Microbial succession during button mushroom (Agaricus bisporus) production evaluated via high-throughput sequencing.

Button mushrooms (Agaricus bisporus), are one of the most widely consumed mushrooms in the world. However, changes within its microbial community as it relates to the use of different raw materials and cultivation methods, as well as potential points of microbial contamination throughout the production process have not been investigated extensively. In the present study, button mushroom cultivation was investigated in each of the four stages (raw materials, composting (phase I, Ⅱ, and Ⅲ), casing, and harvesting), and samples (n = 186) from mushrooms and their related environments were collected from four distinct mushroom-growing farms (A-D) in Korea. Shifts within the bacterial consortium during mushroom production were characterized with 16 S rRNA amplicon sequencing. The succession of bacterial communities on each farm was dependent on the raw material incorporated, aeration, and the farm environment. The dominant phyla of the compost stack at the four farms were Pseudomonadota (56.7%) in farm A, Pseudomonadota (43.3%) in farm B, Bacteroidota (46.0%) in farm C, and Bacillota (62.8%) in farm D. During the Phase Ⅰ, highly heat-resistant microbes, such as those from the phylum Deinococcota (0.6-65.5%) and the families Bacillaceae (1.7-36.3%), Thermaceae (0.1-65.5%), and Limnochordaceae (0.3-30.5%) greatly proliferated. The microbial diversity within compost samples exhibited a marked decline as a result of the proliferation of thermophilic bacteria. In the spawning step, there were considerable increases in Xanthomonadaceae in the pasteurized composts of farms C and D - both of which employed an aeration system. In the harvesting phase, beta diversity correlated strongly between the casing soil layer and pre-harvest mushrooms, as well as between gloves and packaged mushrooms. The results suggest that gloves may be a major source of cross-contamination for packaged mushrooms, highlighting the need for enhanced hygienic practices during the harvesting phase to ensure product safety. These findings contribute to the current understanding of the influence of environmental and adjacent microbiomes on mushroom products to benefit the mushroom industry and relevant stakeholders by ensuring quality production.

Insight into the evolutionary and domesticated history of the most widely cultivated mushroom Agaricus bisporus via mitogenome sequences of 361 global strains.

Agaricus bisporus is the most widely cultivated edible mushroom in the world with a only around three hundred years known history of cultivation. Therefore, it represents an ideal organism not only to investigate the natural evolutionary history but also the understanding on the evolution going back to the early era of domestication. In this study, we generated the mitochondrial genome sequences of 352 A. bisporus strains and 9 strains from 4 closely related species around the world. The population mitogenomic study revealed all A. bisporus strains can be divided into seven clades, and all domesticated cultivars present only in two of those clades. The molecular dating analysis showed this species origin in Europe on 4.6 Ma and we proposed the main dispersal routes. The detailed mitogenome structure studies showed that the insertion of the plasmid-derived dpo gene caused a long fragment (MIR) inversion, and the distributions of the fragments of dpo gene were strictly in correspondence with these seven clades. Our studies also showed A. bisporus population contains 30 intron distribution patterns (IDPs), while all cultivars contain only two IDPs, which clearly exhibit intron loss compared to the others. Either the loss occurred before or after domestication, that could suggest that the change facilitates their adaptation to the cultivated environment.

Insights into the genomic evolution and the alkali tolerance mechanisms of Agaricus sinodeliciosus by comparative genomic and transcriptomic analyses.

Agaricus sinodeliciosus is a rare wild edible mushroom from northwest China, and grows naturally in mild saline-alkali soil, which is also unusual in mushrooms. A. sinodeliciosus represents a potential model organism for explaining saline-alkali tolerance mechanisms and revealing related physiological processes in mushrooms. Here, we provide a high-quality genome of A. sinodeliciosus. Comparative genomic analyses reveal A. sinodeliciosus has numerous changes to its genome organization after a solitary evolutionary history under saline-alkali environments, such as gene family contraction, retrotransposon expansion and rapid evolution of adaptative genes. Our saline and alkali tolerance tests show that mycelium growth and fruit body formation of this species are effected by mild alkalinity. Transcriptomic analyses reveal that genes involved in carbon and nitrogen utilization, cell stability and fruit body formation of A. sinodeliciosus could be activated under mildly alkaline conditions. In particular, the 'starch and sucrose metabolism', 'biosynthesis of amino acids' and 'phenylpropanoid biosynthesis' pathways are important for mildly alkaline tolerance of A. sinodeliciosus. Like plants and arbuscular mycorrhizal fungi, in the rot fungus A. sinodeliciosus, the biosynthesis of intracellular small molecules could be enhanced to counter osmotic and oxidative stresses caused by mild alkalinity, and the biosynthesis of monolignol could be suppressed to increase cell wall infiltrates under mildly alkaline conditions. This research provides an understanding of the genomic evolution and mechanisms of A. sinodeliciosus in tolerance to saline-alkali environments. The A. sinodeliciosus genome constitutes a valuable resource for evolutionary and ecological studies of Agaricus.

Identification of bHLH family genes in Agaricus bisporus and transcriptional regulation of arginine catabolism-related genes by AbbHLH1 after harvest.

Basic helix-loop-helix (bHLH) transcription factors (TFs) are widely distributed in eukaryotes and play an important role in biological growth and development. The identification and functional analyses of bHLH genes/proteins in edible mushrooms (Agaricus bisporus) have yet to be reported. In the present study, we identified 10 putative bHLH members carrying the conserved bHLH domains. Phylogenetic analyses revealed that the 10 AbbHLHs were the closest to sequences of species belonging to 7 different fungal subgroups, which was supported by loop length, intron patterns, and key amino acid residues. The substantial increase after harvest and continuously elevated expression of AbbHLH1 during the development until the disruption of mushroom velum, and the preferential expression in cap and gill tissues suggest the important function of AbbHLH1 in postharvest development of A. bisporus. The relationship of arginine catabolism-related genes with the early stage of postharvest continuing development also was revealed by expression determination. Subcellular localization showed that AbbHLH1 could be localized in nucleus. Importantly, the electrophoretic mobility shift and dual-luciferase reporter assays showed that AbbHLH1 activated the promoters of AbOAT, AbSPDS, and AbSAMDC and suppressed the expression of AbARG, AbUREA, and AbODC, probably for the modulation of arginine catabolism and thus control of postharvest mushroom development. Taken together, the available data provide valuable functional insight into the role of AbbHLH proteins in postharvest mushrooms.

Influence of Agaricus bisporus establishment and fungicidal treatments on casing soil metataxonomy during mushroom cultivation.

The cultivation of edible mushroom is an emerging sector with a potential yet to be discovered. Unlike plants, it is a less developed agriculture where many studies are lacking to optimize the cultivation. In this work we have employed high-throughput techniques by next generation sequencing to screen the microbial structure of casing soil employed in mushroom cultivation (Agaricus bisporus) while sequencing V3-V4 of the 16S rRNA gene for bacteria and the ITS2 region of rRNA for. In addition, the microbiota dynamics and evolution (bacterial and fungal communities) in peat-based casing along the process of incubation of A. bisporus have been studied, while comparing the effect of fungicide treatment (chlorothalonil and metrafenone). Statistically significant changes in populations of bacteria and fungi were observed. Microbial composition differed significantly based on incubation day, changing radically from the original communities in the raw material to a specific microbial composition driven by the A. bisporus mycelium growth. Chlorothalonil treatment seems to delay casing colonization by A. bisporus. Proteobacteria and Bacteroidota appeared as the most dominant bacterial phyla. We observed a great change in the structure of the bacteria populations between day 0 and the following days. Fungi populations changed more gradually, with A. bisporus displacing the rest of the species as the cultivation cycle progresses. A better understanding of the microbial communities in the casing will hopefully allow us to increase the biological efficiency of the crop.

RNA-Seq, Bioinformatic Identification of Potential MicroRNA-like Small RNAs in the Edible Mushroom Agaricus bisporus and Experimental Approach for Their Validation.

Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases.

Metabolomics and transcriptomics unravel the mechanism of browning resistance in Agaricus bisporus.

Agaricus bisporus is widely consumed on the world market. The easy browning of mushroom surface is one of the most intuitive factors affecting consumer purchase. A certain cognition on browning mechanism has been made after years of research. At present, people slow down the browning of mushrooms mainly by improving preservation methods. In addition, breeding is also a reliable way. In the production practice, we have identified some browning-resistant varieties, and we selected a browning-resistant variety to compare with an ordinary variety to reveal the resistance mechanism. Using transcriptomics and metabolomics, the differences in gene expression and metabolite levels were revealed, respectively. The results showed that differentially expressed genes (DEGs) like AbPPO4, AbPPO3 and AbPPO2 were differently expressed and these DEGs were involved in many pathways related to browning. The expression of AbPPO expression play an important role in the browning of A. bisporus and multiple PPO family members are involved in the regulation of browning. However, the resistance to browning cannot be judged only by the expression level of AbPPOs. For metabolomics, most of the different metabolites were organic acids. These organic acids had a higher level in anti-browning (BT) than easy-browning varieties (BS), although the profile was very heterogeneous. On the contrary, the content of trehalose in BS was significantly higher than that in BT. Higher organic acids decreased pH and further inhibited PPO activity. In addition, the BS had a higher content of trehalose, which might play roles in maintaining PPO activity. The difference of browning resistance between BS and BT is mainly due to the differential regulation mechanism of PPO.

Bacterial Community Patterns in the Agaricus bisporus Cultivation System, from Compost Raw Materials to Mushroom Caps.

Different from other fungal species that can be largely cultivated in 'axenic conditions' using plant material (e.g., species of Lentinula and Pleurotus in 'sterile' straw-based substrate), the commercial Agaricus bisporus cultivation system relies heavily on ecological relationships with a broad range of microorganisms present in the system (compost and casing). Since the A. bisporus cultivation system consists of a microbial manipulation process, it is important to know the microbial community dynamics during the entire cultivation cycle to design further studies and/or crop management strategies to optimize this system. To capture the bacterial community 'flow' from compost raw materials to the casing to the formation and maturation of mushroom caps, community snapshots were generated by direct DNA recovery (amplicon sequencing). The 'bacterial community flow' revealed that compost, casing and mushrooms represent different niches for bacteria present in the cultivation system, but at the same time, a bacterial exchange between microenvironments can occur for a portion of the community. Within each microenvironment, compost showed intense bacterial populational dynamics, probably due to the environmental changes imposed by composting conditions. In casing, the colonization of A. bisporus appeared, to reshape the native bacterial community which later, with some other members present in compost, becomes the core community in mushroom caps. The current bacterial survey along with previous results provides more cues of specific bacteria groups that can be in association with A. bisporus development and health.

Transmission of mushroom virus X and the impact of virus infection on the transcriptomes and proteomes of different strains of Agaricus bisporus.

Cultivation of Agaricus bisporus is a large horticultural industry for many countries worldwide, where a single variety is almost grown exclusively. Mushroom virus X (MVX), a complex of multiple positive-sense single stranded RNA (ss(+)RNA) viruses, is a major pathogen of typical A. bisporus crops. MVX can manifest a variety of symptoms in crops and is highly infective and difficult to eradicate once established in host mycelium. Currently our knowledge regarding the molecular response of A. bisporus fruit bodies to MVX infection is limited. In order to study the response of different A. bisporus strains with different susceptibilities to MVX, we designed a model system to evaluate the in-vitro transmission of viruses in A. bisporus hyphae over a time-course, at two crucial phases in the crop cycle. The symptom expression of MVX in these varieties and the transcriptomic and proteomic response of fruit bodies to MVX-infection were examined. Transmission studies revealed the high potential of MVX to spread to uninfected mycelium yet not into the fruit bodies of certain strains in a crop. MVX affected colour and quality of multiple fruit bodies. Gene expression is significantly altered in all strains and between times of inoculation in the crop. Genes related to stress responses displayed differential expression. Proteomic responses revealed restriction of cellular signalling and vesicle transport in infected fruit bodies. This in-depth analysis examining many factors relevant to MVX infection in different A. bisporus strains, will provide key insights into host responses for this commercially important food crop.

Mating-Type Locus Organization and Mating-Type Chromosome Differentiation in the Bipolar Edible Button Mushroom Agaricus bisporus.

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and ß-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the ß-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between ß-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

Three Mycogone Species, Including a New Species, Cause Wet Bubble Disease of Agaricus bisporus in China.

White button mushroom, Agaricus bisporus (Lange) Imbach, is the most extensively cultivated and edible mushroom worldwide. The production of A. bisporus is commonly affected by wet bubble disease (WBD), imposing a significant economic burden in China. Although studies have shown that this disease is caused by fungi of the Mycogone genus, the pathogen has not been fully characterized. In this study, 802 samples of diseased fruiting bodies of A. bisporus were collected from nine major mushroom-cultivating provinces in China, yielding a total of 586 Mycogone isolates. The morphologic characteristics of these isolates were observed and compared, and multilocus phylogenetic analyses (internal transcribed spacer [ITS], ACT, TEF1-α, TUB, RPB2, and large ribosomal subunit [LSU]) were performed on the selected representative isolates. Three Mycogone species were identified: a new species, M. xinjiangensis; M. perniciosa; and M. rosea. Mycogone rosea was the first ever reported in China. Furthermore, M. rosea was found to be the most prevalent species (54.95% of all isolates) in all the sampled areas, except in Hubei and Xinjiang, followed by M. perniciosa (39.93%) and M. xinjiangensis (5.12%). Pathogenicity tests on the fruiting body and mushroom bed substantiated Koch's postulates by the development of mildly different symptoms after inoculation with each species. This study, therefore, enhances our knowledge of the species associated with WBD in A. bisporus and provides useful insights for preventing WBD and allied diseases.

Anthocyanin extract from Lycium ruthenicum enhanced production of biomass and polysaccharides during submerged fermentation of Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC) is a wild edible fungus uniquely found in the Tibet Plateau. ABSC is rich in polysaccharides that are considered biologically active. This study aimed to determine the feasibility of enhancing exopolysaccharide (EPS) production by ABSC in shake flask culture by supplementing the fermentation medium with anthocyanin extract. Different concentrations of Lycium ruthenicum Murr. (LRM) anthocyanin crude extract were tested on ABSC fermentation. The activity of phosphoglucose isomerase (PGI), phosphoglucose mutase (PGM), and phosphomannose isomerase (PMI), enzymes presumably involved in EPS synthesis by ABSC, was determined. ABSC transcriptomic profile in response to the presence of anthocyanins during fermentation was also investigated. LRM anthocyanin crude extract (0.06 mg/mL) was most effective in increasing EPS content and mycelial biomass (by 208.10% and 105.30%, respectively, P < 0.01). The activity of PGI, PGM, and PMI was increased in a medium where LRM anthocyanin extract and its main components (proanthocyanidins and petunia anthocyanin) were added. RNA-Seq analysis showed that 349 genes of ABSC were differentially expressed during fermentation in the medium containing anthocyanin extract of LRM; 93 genes were up-regulated and 256 genes down-regulated. From gene ontology enrichment analysis, differentially expressed genes were mostly assigned to carbohydrate metabolism and signal transduction categories. Collectively, LRM anthocyanins extract positively affected EPS production and mycelial biomass during ABSC fermentation. Our study provides a novel strategy for improving EPS production and mycelial growth during ABSC liquid submerged fermentation.

An overview of Agaricus section Hondenses and Agaricus section Xanthodermatei with description of eight new species from Pakistan.

In a recent revision of the genus Agaricus, A. section Xanthodermatei was split into two sections A. sect. Hondenses and A. sect. Xanthodermatei. Our objectives were to investigate the species diversity of both sections in Pakistan and to give an overview of the major clades. Phylogenetic analyses based on the combined nucLSU, ITS and TEF1 dataset from 35 specimens of both sections revealed three major clades. Analyses based on ITS dataset and 106 specimens, including 33 from Pakistan, reveal eight new species and one new record species. These nine species are described in detail. It is noteworthy that intraspecific variability as well as interspecific variability between closely related species were very low in ITS sequences in many cases. In the case of the two new species A. xanthochromaticus and A. griseovariegatus, TEF1 sequence data were much more efficient than ITS to distinguish these species from each other. The other new species are A. atroumbonatus, A. fumidicolor, A. macropeplus, A. parviniveus, A. swaticus and A. bambusetorum. The latter is the only new species of A. sect. Hondenses in which it is morphologically atypical and also the unique (sub)tropical species. Agaricus gregariomyces is recorded for the first time in Pakistan. In addition, brief descriptions are provided not only for A. bisporiticus, A. endoxanthus and A. punjabensis, which are reported again in Pakistan, but also for A. californicus, which is reported for the first time in Spain and outside North America. In total 12 species of both sections were reported in Pakistan and half of them were from subtropical climatic areas, underlining the contribution of the climatic diversity to the high species richness in this country.